HLA Biology
Human leukocyte antigens (HLAs), i.e. the human major histocompatibility complex (hMHC) molecules, are encoded by a cluster of MHC genes in a continuous region on human chromosome 6 (21.31p). The major function of HLA proteins are cell-cell recognition and self-non-self discrimination in the immune system. There are two major groups of HLA, HLA class I and HLA class II. HLA class I molecules are expressed on the surface of most nucleated cells and present peptides generated mostly from an endogenous route and sometimes from an exogenous route to CD8+ T cells to stimulate their functions. The stimulated CD8+ T cells can recognize infected cells or tumor cells and kill them directly through cell cytotoxicity. Besides presenting peptides for T cell recognition, MHC class I molecules can inhibit NK cell activation by interacting with inhibitory receptors, such as KIR-2DL and KIR-3DL, on NK cell surface.
For MHC class II molecules, they can be expressed constitutively only on antigen presenting cells (APCs), such as B cells and dendritic cells, and present exogenous antigens to CD4+ helper T cells. The stimulated CD4+ T cells can either help cell-mediated immunity or help antibody production.
Generally, a fully-formed MHC class I complex has three components, a 45 kDa heavy chain, a 12 kDa β2-microglobulin (β2m) and an 8-10 amino acid peptide. In heavy chain, there are 3 extracellular domains (i.e., α1, α2 and α3), a hydrophobic transmembrane domain and a hydrophilic cytoplasmic domain. In an intact HLA class I complex, the α1 and α2 domains of heavy chain form the cleft for peptide binding and the α3 domain binds to β2m and provides the species specificity for CD8 binding.
Antigen Processing Machinery (APM)
The assembly of MHC class I molecules relies on a number of chaperon proteins collectively named antigen processing machinery (APM) components in the endoplasmic reticulum (ER) lumen. The peptide presented by MHC class 1 molecules can come from the endogenous proteins produced by viruses living in the infected cells or by tumor cells. In the cytosol, these endogenous proteins are tagged with ubiquitin and cleaved into small peptides by proteasomes and/or immunoproteasomes in cells. The cleaved peptides will associate with transporters associated with antigen processing (TAP), which is an ATP-binding-cassette transporter complex composed of 70 kDa TAP1 and 74 kDa TAP2, and are transported into ER. In the ER lumen, the MHC class I heavy chain is partially folded with the chaperon protein, calnexin and BiP, initially. Subsequently, the MHC class I heavy chain will assemble with β2m, stabilized by calreticulin and ERp57, and then connected to TAP through TAP-associated glycoprotein tapasin. Once the appropriate peptide, coming from the TAP, binds to the MHC class I molecule, calreticulin, ERp57, tapasin and TAP will dissociate from MHC class I molecule. Finally, the intact MHC class I molecule leaves the ER and is transported through endomembrane system to cell membrane for CD8+ T cell recognition.
Recombinant MHC Class I Molecules
Construction of recombinant MHC class I molecules produced by E. coli system was developed in 1992. Later, the first structure of recombinant MHC class I molecules was released. As previously mentioned, an intact MHC class I contains three components: a heavy chain, a β2m and an 8-10 amino acid peptide. The three components are non-covalently associated. For constructing the intact MHC complex, the heavy chain and β2m should be produced separately in the E. coli system and the small peptide needs to be synthesized artificially which is expensive, low-yielding and time-consuming.